Computer-based design and screening of molecules using DNA interactions

ABSTRACT

It has been discovered that the degree of hormonal activity of candidate ligands correlates better with degree of fit into DNA than with the strength of receptor binding, and that the receptors in the steroid/thyroid hormone/vitamin A and D family alter the physiochemical properties of DNA and in concert with other transcription factors facilitate insertion of the ligand into DNA. As a result, the magnitude of the response is a function of the structure of the ligand as it related to insertion and fit into the DNA and the specificity of the response is a function of the stereochemistry of the receptor through binding to both the ligand and to the DNA. Based on these discoveries, a method is described herein for identifying drugs having increased activity as compared with the natural ligand for receptors such as the estrogenic receptors.

This is a divisional of Application Ser. No. 08/369,779, filed Nov. 28,1994, U.S. Pat. No. 5,705,335, which is a continuation in part ofapplication Ser. No. 08/158,689, filed Nov. 26, 1993, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to rational drug design, inparticular design of biologically active molecules using pharmacophoresprepared according to the present invention.

BACKGROUND OF THE INVENTION

Why certain chemical structures and not others are present in nature hasbeen a recurring question raised by scientists since the first organicnatural products were characterized. Of equal interest has beenelucidating what structural features within any given class of organicmolecules are responsible for biological activity. Historically, thelack of satisfactory answers to both questions has relegated thedevelopment of biologically active molecules either to serendipity or toexhaustive synthesis and biological testing of large numbers ofcompounds. This frustration is particularly evident in thepharmaceutical industry where the development of drug agonists andantagonists is often time consuming, tedious and expensive.

This picture is beginning to change as more information is derived frommodern molecular modeling techniques including characterization of theactive sites in enzymes and the ligand binding sites in receptors. Overthe past 15 years, another approach has emerged based upon a series ofdiscoveries made with molecular models, wherein biologically activesmall molecules have been found to possess complementary stereochemicalrelationships with gene structure. This approach was first described inU.S. Pat. No. 4,461,619 to Hendry, et al., which is incorporated hereinby reference. This simple molecular modeling technology was developedfrom observations first reported in 1977 of structural relationshipsbetween small molecules and nucleic acids, as described by Hendry, etal., J, Steroid Biochem. Molec. Biol. 42:659-670 (1992); Copland, etal., J. Steroid Biochem. Molec. Biol. 46:451-462 (1993); Hendry andMahesh, J. Steroid Biochem. Molec. Biol. 41:647-651 (1992); Witham andHendry, J. Theor. Biol. 155:55-67 (1992); Hendry and Mahesh, J. SteroidBiochem. Molec. Biol. 39:133-146 (1991); Hendry, J. Steroid Biochem.31:493-523 (1988); Lehner, et al., Molec. Endocrinol. 1:377-387 (1987);Hendry, et al., J. Steroid Biochem. 24:843-852 (1986); Uberoi, et al.,Steroids 45:325-340 (1985); Bransome, et al., J. Theor. Biol. 112:97-108(1985); Hendry, et al., Proc. Natl. Acad. Sci. USA 78:7440-7444 (1981);and Hendry, et al., Perspect. Biol. Med. 27:623-651 (1984), all of whichare incorporated herein by reference.

The essential ingredient of all genes is a single, well defined polymer,deoxyribonucleic acid (DNA). DNA is a remarkably uncomplicated moleculecomposed of recurring sugar-phosphate units attached to one of fourpossible bases: adenine (A), thymine (T), cytosine (C) or guanine (G).The simplicity of gene structure is further evident in the Watson andCrick base pairing scheme of double-stranded DNA (A with T and C withG), and the helical chirality (handedness) dictated by the absoluteconfiguration of the sugar D-deoxyribose. Gene structure couldconceivably be composed of many other chemical units, for example, othersugar stereoisomers such as L-deoxyribose or sugar homologs related toD-glucose.

The products of gene structure, proteins, are also simple, ubiquitousmolecules. Nature limits the structure of proteins by constructing themfrom only twenty basic units, the amino acids; protein chirality isconstrained by the absolute L-configuration of the amino acids. As inthe case of nucleic acid subunits, a wide range of structuralalternatives are possible for protein amino acids. Examples includechanges in the chirality of a given amino acid side chain (e.g.,D-isoleucine), rearrangements in the pattern of atoms (e.g., the t-butylisomer of isoleucine) or the addition of atoms (e.g., pipecolic acid, ahomologue of proline).

Structural constraints are also evident in the stereochemistry of lowmolecular weight natural products. Particularly conspicuous arelimitations imposed by nature on the number, size, shape, elementalcomposition, and chirality of biologically active small molecules. Forexample, the pervasive neurotransmitters histamine and serotonin areunique in that alternative structures with changes in the position orcomposition of heteroatoms and/or ring patterns generally do not existin nature. Similarly, many small molecular weight hormones are few innumber, have recurring structural patterns and possess a single absolutechirality.

The source of the pervasive occurrence of physicochemical constraints onthe structure of naturally occurring small molecules lies directly inthe structure of the proteins which govern both their biosynthesis andbioactivity, i.e., enzymes and receptors, respectively. Ultimately,however, this stereochemical information is contained in the genes.According to the basic tenants of molecular biology, the information inDNA is replicated with remarkable precision and fidelity into newlysynthesized DNA. It is also transcribed into RNA and subsequentlytranslated into protein.

This scenario, however, presents an apparent paradox. While the genetictemplate ultimately directs which proteins and small molecules aresynthesized, as well as which proteins and small molecules will interactwith each other, the undirectional flow of genetic information duringtranslation suggests that DNA structure performs this function withoutrecognizing the structure of the small molecule. With few exceptions,such as certain antibiotics which bind directly to DNA and blocktranscription, small molecules are not considered to recognize orinteract with the genetic template. Moreover, the structures of themolecules that are biosynthesized are thought to be unrelated to thestructure of the genes.

In the initial search for structural relationships between biologicallyactive natural products and DNA, it became apparent that thetwo-dimensional structures of DNA base pairs were analogous to manyclasses of small molecules, including gibberellic acid, a phytohormone;benzo a! pyrene oxide, a carcinogen; the prostaglandin PGE₂ ; morphine,a narcotic; estradiol, a hormone; riboflavin, vitamin B₁₂ ; serotonin, aneurotransmitter; and actinomycin, an antibiotic. In addition tosimilarities in size and shape, numerous small molecules containeddonor/acceptor functional groups at locations where hydrogen bondsoccurred between the base pairs. When overlaid on the base pairs, somecompounds, such as the plant hormone gibberellic acid, the steroidhormone estradiol, and prostaglandins, contained heteroatoms separatedby internuclear distances similar to that of phosphate oxygens onadjacent strands of double-stranded DNA. This was particularly evidentin functional groups attached at the 3 and 17β positions of thesteroids.

Using three dimensional Corey-Pauling-Koltun (CPK) space filling models,it became apparent that there were spaces between base pairs inpartially unwound DNA that could accommodate a variety of smallmolecules. For example, estradiol could be inserted between base pairsin DNA, and the hydroxyl groups at 3 and 17β of estradiol werepositioned such that they could form hydrogen bonds to phosphate oxygenson adjacent strands of DNA. Other steroids, including testosterone andprogesterone, were also capable of stereochemical insertion between basepairs. In each case, complementary donor/acceptor linkages could beformed and the steroid conformed well to the topography of the doublehelix. Attempts to insert any of the non-naturally occurring steroidenantiomers into DNA resulted in poor fit in that donor/acceptorlinkages were strained or could not form, and/or the overall shape ofthe molecules was incompatible with the helical topography of the DNA.

Certain synthetic compounds with hormonal activity can also beaccommodated within the DNA; in many cases, the fit of syntheticcompounds such as diethylstilbestrol mimicked that of the naturalhormone. In addition to mammalian steroids, prostaglandins, the insecthormone ecdysone and several phytohormones were also capable ofstereochemical insertion and "recognition" by the double helix. In thecase of the plant hormone gibberellic acid, four stereospecific hydrogenbonds could be formed to donor/acceptor positions on the DNA. As withthe steroids, only the naturally occurring enantiomer of gibberellicacid conformed to the topography of the double helix.

One conclusion drawn from these studies is that certain chemical shapes,coupled with heteroatom positioning compatible with that of thephosphate backbone of DNA and hydrogen bond positions of the base-pairtemplate, potentiate partial or complete recognition betweenbiologically active molecules and DNA.

While it was possible to form complexes between DNA and a variety ofmolecules, amino acids did not initially show any clear accommodation tothe space between base pairs. Certain compounds derived from aminoacids, for example, neurotransmitters, fit into related sites.

These relationships have been described as a stereochemical logicassociated with gene structure. The stereochemical logic is defined asthose unique features of nucleic acid structure which ultimately dictateconstraints on molecular structure, function, metabolism, and biologicactivity.

The use of molecular modeling as a tool to study organic structure hasdramatically increased due to the advent of computer graphics. Not onlyis it possible to view molecules on computer screens in three dimensionsbut it is also feasible to examine the interactions of ligands withvarious macromolecules such as enzymes and receptors, as reviewed byBorman, Chem. Eng. News 70:18-26 (1992). An almost baffling array ofsoftware and hardware is now available and virtually all majorpharmaceutical companies have computer modeling groups which are devotedto drug design.

Modern methods of drug design include studies which focus on the bindingof a molecule to a protein such as a polypeptide ligand for a receptor,or a steroid such as an estrogen or progesterone for a receptor.Similarly, drugs can be designed based upon the interaction ofsubstrates with various enzymes. For the most part, however, bindingsites in proteins have been difficult to characterize. There are manysituations where other mechanisms must be involved to explain thefeedback between protein regulation and regulation of gene expression.

What is needed is a method for accurately predicting the biologicalactivity of a given compound. The method should be easy to perform andshould be able to predict both agonist and antagonist activity.

SUMMARY OF THE INVENTION

The present invention is a method for idenitifying biological activityof molecules using pharmacophores. According to the present invention,molecules are screened by determining the degree of "fit" in thepharmacophore.

The method according to the present invention can be used to identifydrugs having increased biological activity or which have usefulness asantagonists or agonists, including, for example, estrogens andanti-estrogens. This method can also be used for the following: topredict the fit of compounds into nucleic acids, especially DNA; topredict the bioactivity of compounds, to screen compounds for toxicity;to design chemical groups to add to specific sites on molecules tofacilitate metabolism or render the drug an agonist or antagonist; andto create molecules that mimic the activity of the DNA binding regionsof receptors.

The present invention also includes pharmacophores and the method ofproducing the pharmacophores and the use of the pharmacophores inpredicting biological activity of a given compound. The presentinvention also includes the design of biologically active moleculesusing the pharmacophore.

It is therefore an object of the present invention to provide a methodwhich can be used to design a biologically active molecule.

It is another object of the present invention to provide a method toscreen and/or evaluate existing compounds for toxicological activity.

Still another object of the present invention is to provide a method topredict the toxicity of compounds.

Another object of the present invention is to provide a method topredict the toxicity of compounds for specific organs, tissues, andcells.

Yet another object of the present invention is to provide a method todesign compounds that will have particular types of biologicalactivities, including, but not limited to, hormonal, neurotransmitter,metabolic, genetic, immunologic, pathologic, toxic, and anti-mitoticactivities.

Still another object of the present invention is to provide a method topredict the bioactivity of compounds including, but not limited toestrogenic, anti-estrogenic, androgenic, anti-androgenic,progestational, anti-progestational, mineralocorticoid, retinoid,vitamin D like, thyroid, and glucocorticoid bioactivities.

Yet another object of the present invention is to provide a method tocreate pharmacophores that can be used to design compounds such asdrugs, hormones, neurotransmitters, agonists and antagonists moreefficiently and economically.

It is another object of the present invention to provide a method tocreate receptor pharmacophores that are molecular models of the portionsof receptor molecules that bind to nucleic acids.

Yet another object of the present invention is to provide a receptorpharmacophore that can be used to design molecules that bind to nucleicacids with different affinity than the receptor.

Yet another object of the present invention is to provide apharmacophore that represents the three dimensional arrangement ofsolvent molecules around the ligand pharmacophore that binds to nucleicacids.

Another object of the present invention is to provide a pharmacophorethat represents the three dimensional arrangement of solvent moleculesaround the receptor pharmacophore that binds to nucleic acids.

It is yet another object of the present invention to provide a method tocreate a pharmacophore that is a three dimensional model of the nucleicacid binding domain of the receptor and of the ligand molecule thatbinds to the receptor and interacts with the nucleic acid at a differentsite.

Another object of the present invention is to provide a pharmacophorethat represents the three dimensional arrangement of molecules that canbe attached to other pharmacophores to modify their biological activity.

Still another object of the present invention is to provide apharmacophore that represents the three dimensional arrangement ofmolecules that can be attached to other pharmacophores in order todesign sites for enzymatic cleavage.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of necessary fee

FIG. 1 is a schematic of a cavity in DNA and the numbered sites whichaccommodate steroid/thyroid/vitamin A and D ligands: testosterone (1,7);progesterone (2,7); aldosterone (2,5,7 and 9); cortisol (2,3,4,5, and7); estradiol (1 and 6); triiodothyronine (T₃) (1 and 6); retinoic acid(6); and 1,25- (OH)₂ vitamin D₃ (1 and 6); these are divided into twogroups based on their interaction with either site 6 or 7.

FIGS. 2A-2C depict a computer generated space filling stereo view of theDNA cavity (A), which fits active estrogens oriented by energycalculations into the DNA cavity (B), whereas (C) shows the combinedactive surface of estrogens removed from the cavity in DNA that is usedto construct the pharmacophore.

FIGS. 3A-3D depict a volume contour map (yellow) in stereo with dummyatoms (magenta) surrounding the active molecules which were used in theconstruction of the pharmacophore (A); the empty pharmacophore (B); fitof the highly active estrogen3,11β,17β-trihydroxy-7α-methylestra-1,3,5(10)-triene 11-nitrate ester(hereinafter 7α-methylestradiol-11β-nitrate ester reported in Peters etal., J. Med. Chem. 32:2306-2310 (1989)) which is accommodated completelywithin the pharmacophore (C); and poor fit of the inactive estrogen9β-estradiol which extends appreciably beyond the surface of thepharmacophore (D).

FIGS. 4A-4E depict a stereo view of volume maps (green mesh) and dummyatoms (magenta) for pharmacophores for estrogen (A), androgen (B),thyroid (C), anti-estrogen (D), and toxicity (E). Dummy atoms are notpresented with the toxicity pharmacophore volume map.

FIGS. 5A-5E, left column, shows the volume maps (green mesh) and dummyatoms (magenta) for pharmacophores for estrogen (A), androgen (B),thyroid (C), anti-estrogen (D), and toxicity (E). The right columnpresents an orthogonal view (90 degree rotation) of the correspondingimages in the left column.

FIG. 6 demonstrates the relationship between the degree of fit ofvarious compounds to the estrogen pharmacophore and the relativeuterotropic (estrogenic) activity.

FIGS. 7a-7c show the chemical structures of three separate molecules,SGI 100, SGI 101, and SGI 102 designed with pharmacophore technology.

DETAILED DESCRIPTION OF THE INVENTION

Many natural products have structures that exhibit stereochemicalcomplementarity with nucleic acids, including amino acids,phytohormones, cyclic nucleotides, prostaglandins, insect hormones,steroid hormones, neurotransmitters, sugars, peptide hormones, thyroidhormones, pheromones, and vitamins. A striking example is thecyclopentanophenanthrene motif repeated in all classes of mammaliansteroid hormones, such as estrogen and progesterone. Another example isthe kaurene nucleus containing eight chiral centers which is evident inthe gibberellin class of plant hormones.

There are many ways to examine the stereochemistry of variousconfigurations and conformations of nucleic acids. For example, Silasticpolymer models can be constructed based upon computer derived spacefilling x-ray coordinates to reflect the stereochemistry of partiallyunwound DNA/RNA complexes, RNA--RNA complexes, ent-DNA (mirror image ofDNA made with L-deoxyribose), and apurinic/apyrimidinic sites in DNA.These cavities reveal a sequence specificity in the fit of manymolecules. The apurinic/apyrimidinic sites accommodate amino acidsaccording to the known genetic code. The plant hormone gibberellic acidfits best into the partially unwound site 5'-dTdA-3', 5'-dTdA-3';members of the mammalian steroid/thyroid hormone superfamily fit bestinto 5'dTdG-3', 5'-dCdA-3'. Each class of mammalian hormone forms uniquestereospecific donor- acceptor hydrogen bonds with DNA. The capacity tofit within these cavities in the manner of the index biologically activemolecule correlates with the degree of biologic activity. It is notpossible to fit chiral naturally occurring molecules into ent-DNA.However, ent-DNA accommodates the biologic unknown chiral enantiomers,such as ent-progesterone.

According to the present invention, computer modeling is used to examinethe relationships between compounds and their fit in helical DNA.Although described herein with reference to double stranded, helicalDNA, many of the same principles are applicable to double stranded RNA,and/or to RNA-DNA hybrids. Unless otherwise specified, double strandedRNA and DNA are to be considered equivalents as used herein. Computermodeling can be used to view the interactions of molecules as well as tomeasure the energy of a given interaction. While a variety of softwarepackages are available for computer modeling of molecules, a preferredsoftware package is Sybyl software (version 6.03; Tripos Associates, St.Louis, Mo.) for measuring the docking of various small molecular weightligands into DNA. In the examples described herein, the software is runon a Silicon Graphics Indigo Extreme equipped with hardware stereo,i.e., Crystal Eyes (StereoGraphics, San Rafael, Calif.). Structures ofsmall molecules are obtained via: the Cambridge CrystallographicDatabase, Lanfield Rd., Cambridge, England; construction with theConcord program or from fragment libraries and/or modifications ofexisting x-ray structures followed by energy minimization. All energycalculations are made using the Sybyl force field with a 1.2 Å van derWaals parameter for hydrogen, as described by Hendry, et al., J. SteroidBiochem. Molec. Biol. 42:659-670 (1992) and Hendry, et al., J. SteroidBiochem. Molec. Biol. 39:133-146 (1991). Charges are calculated usingthe Gasteiger-Huckel method to include σ and π bonding. Partiallyunwound DNA cavities of various double stranded dinucleotide sequencesare constructed from the Watson and Crick canonical B form of DNA bytwisting each of the fourteen torsional angles along the sugar-phosphatebackbone while maintaining the structural integrity of hydrogen bondsbetween the base pairs. The 3'-endo deoxyribose conformation of thesugars is employed and Kollman charges are calculated. Within theseconstraints, attempts are made to adjust the degree of unwinding and theresulting degree of separation of the base pairs to accommodate thewidth of various ligands

Each ligand is inserted into the cavity in DNA using van der Waals dot,mesh, and space filling surfaces in stereo to guide the dockingprocedure and minimize steric strain. The oxygens of the phosphategroups are permitted to act either as proton donors or acceptors andoriented to maximize the fit of any given ligand. The docking of themolecules is repeated several times. The distances between heteroatomsare monitored interactively to optimize the direction and distances ofpotential hydrogen bonds. While evaluating fit of compounds in a givenhormone class, attempts are made to insert all candidate ligands intothe DNA sequence with donor/acceptor linkages common to the hormone.Donor/acceptor relationships are further maximized by appropriateorientation of functional groups on the ligands, e.g., by adjusting theconformation of each structure to best mimic the fit of the hormone.

Van der Waals interactions of the candidate ligands are optimized withthe DNA surface. The force field is used to calculate the relative fitof each ligand by assessing the optimum favorable energy changeresulting from docking the ligand. Each ligand is docked into the DNAand the steric fit is calculated from the change in van der Waalsenergy; the hydrogen bonding fit is calculated from the change inelectrostatic energy using charges on donor hydrogens and acceptorheteroatoms. The energy changes are assessed for each ligand. Thegreater the negative energy change upon insertion of a given ligand intoDNA, the more favorable the fit and the more stable the complex. Optimaldocking is determined when no further increase in fit is observed. Themost favorable change in energy is selected to compare the relative fitof each molecule. The overall fit of each ligand is assessed by addingthe change in kcal of the van der Waals and electrostatic energies andnormalizing the fit to that of the parent hormone.

Complementarity of Biologically Active Structures

Although not wanting to bound by the following hypothesis, it isbelieved that the mechanism of action of hormones in the steroid/thyroidsuperfamily where ligand bound receptor is known to regulate hormoneresponsive genes is mediated by physically inserting small moleculesinto DNA. The prior art teaches that the ligand causes a specificconformational change in the receptor protein which in turn contacts theDNA resulting in gene regulation. In contrasts according to the presentinvention, there is a receptor-mediated insertion of the ligand intoDNA. This is consistent with the widely reported lack of correlationbetween hormonal activity and receptor binding for estrogenic steroids.According to Brooks, et al., Recent Advances in Steroid Hormone Action,Moudgil, V. K. (ed) 443-466 (Walther de Gruyter, NY 1987), who made anextensive study of estrogen structure-function relationships, receptorbinding is essential for target-cell responses elicited by the steroids.However, the affinity of altered estrogens is not directly related tothe character or extent of the response. At the same time, the bindingof estrogens and other steroids to DNA in the absence of receptor hasbeen observed to be weak by numerous investigators. These includestudies of the flat phytoestrogen coumestrol which might be expected toeasily slide between base pairs. In short, the binding of the steroidindependently to either the receptor or the DNA does not appear to besufficient to fully explain hormonal response.

Several pieces of evidence indicate that certain molecules, such assteroids, that have been shown to fit remarkably well between base pairsin DNA may elicit strong biological responses albeit through relativelyweak interactions with DNA. Support for this concept is based on invitro and in vivo experimental data and the energy calculationsdemonstrating a correlation between fit of estrogens into DNA anduterotropic activity, for example, studies of a new class of antitumoragents which have led to the discovery of drugs that are potent but actby binding weakly to DNA via intercalation, reported by Lee, et al., JMed. Chem. 35:258-266 (1992). Additional support that binding toreceptors alone is insufficient to explain activity is provided by theobservation of poor binding of a potent estrogen analog,11β-acetoxyestradiol, which is considerably more active than estradiol.

In general, given that degree of hormonal activity does not alwayscorrelate well with receptor binding but does correlate with fit intoDNA, it follows that the mode of action of steroids involves a stage(s)in which the ligand is recognized by both the receptor and the DNA. Thisconclusion has also been reached in studies of anti-androgens.

It is contemplated as part of the present invention that binding of thesteroid to its receptor serves as a means to recognize the general classof hormone (e.g., estrogen versus androgen) whereas the type and degreeof the fit of the steroid in the DNA is largely responsible forgoverning the magnitude of the biologic response. The steroid alone isincapable of proper insertion into DNA either in vivo or in vitrowithout the receptor and thus would be unable to generate a fullhormonal response without the receptor A potentially critical role forthe receptor upon binding to DNA possibly in concert with othertranscription factors, is to regulate the physicochemical properties ofthe site in DNA to permit insertion of the steroid, including the degreeof unwinding, the capacity of heteroatoms to act as either donors oracceptors, and the pattern and degree of solvation. This is furthersupported by the decrease in the surface hydrophobicity of the receptorupon binding estrogens and antiestrogens as well as phosphorylationwhich enhances binding of the estrogen receptor to specific DNAsequences, reported by Denton, et al., J. Biol. Chem. 267:7263-7268(1992).

The process of a receptor-mediated insertion of steroids into DNApresumably involves several steps. For example, in the case ofestrogens, the initial contact of the DNA by the steroid-receptorcomplex could involve a partially exposed D ring of the steroid with theA ring still attached to the receptor. Analysis of receptor binding datasupports this possibility. Stereospecific recognition of the DNA bypartial insertion and hydrogen bonding of the 17β-hydroxyl of thesteroid D ring with the 5'-dTdG-3' strand could be followed by completeinsertion and the recognition within the double helix manifest by thelinkage of both stereospecific hydrogen bonds. The weak binding observedfor the steroids with DNA suggests that the steroid/DNA complex might beshort lived and rapidly reversible. Certain estrogen antagonists whichare accommodated differently in DNA may form longer lived interactionswithin the site.

There are numerous possible scenarios and implications of receptormediated binding of ligands to nucleic acids. For example, the lack ofeffectiveness of certain anti-estrogens in tissues which lack estrogenreceptor might be due in part by the inability of the antagonist to betransported to the DNA obviating the insertion of the ligand. Mutationof the site which accommodates the estrogens would also result inimproper recognition of the ligand and would be predicted to no longerpermit the gene to be properly regulated either by agonists orantagonists. There might be multiple levels at which a given compoundmight act, as suggested by the observation that cavities in doublestranded RNA and RNA-DNA hybrids can accommodate various ligands, forexample, estradiol in 5'-rUrG-3', 5'-dCdA-3'.

Molecular Modeling

Molecular modeling was performed with Sybyl/Mendyl 5.4 (TriposAssociates, St. Louis, Mo.) using an Evans and Sutherland PS390 graphicscomputer equipped with a stereographic viewer. Structures ofpiperidinedione ligands were provided via construction with the Concordprogram or from fragment libraries followed by energy minimization.Energy calculations were made with Sybyl/Mendyl force field and a 1.2Avan der Waals parameter for hydrogen. Charges were calculated using theGasteigner-Huckel method which includes σ and π bonding. Partiallyunwound DNA was constructed with 3'-endo deoxyribose conformation andKollman charges. This method has been described by Hendry, et al., J.Steroid Biochem. Molec. Biol. 42:659-670 (1992); Hendry, et al., J.Steroid Biochem. Molec. Biol. 39:133-146 (1991), and Hendry et al., J.Steroid. Biochem. Molec. Biol. 49: No. 4-6, pp. 269-280 (1994) theteachings of which are hereby incorporated by reference in theirentirety.

The ligands were inserted into the cavity in DNA using van der Waals dotsurfaces and the stereoviewer to guide the docking procedure andminimize any obvious steric strain. The distances between heteroatomswere monitored interactively to optimize the direction and distances ofpotential hydrogen bonds. Donor/acceptor relationships were furthermaximized by appropriate orientation of functional groups on theligands, e.g., by adjusting the conformation of each structure. Attemptswere made to optimize van der Waals interactions of the candidateligands with the DNA surfaces. The force field was used to assess therelative fit of each ligand by quantitating the optimum favorable energychange resulting from docking the ligand. Steric fit was calculated fromthe change in van der Waals energy, the hydrogen bonding fit wascalculated from the change in electrostatic energy using charges ondonor hydrogens and acceptor oxygens. The greater the negative energychange upon insertion of a given ligand into DNA, the more favorable thefit and the more stable the complex. Docking was completed when nofurther increase in fit was observed. The most favorable change inenergy was selected to compare the relative fit of each molecule. Theoverall fit of each ligand was assessed by adding the change in kcal ofthe van der Waals and electrostatic energies and normalizing the valueto that of the best fitting molecule (100%). It should be noted thatwhile the energies reported here were derived from widely used forcefield calculations, they were not empirically derived. Thus, theabsolute values in kcal do not have independent experimentalsignificance. At the same time, they are valuable indicators of therelative degree of fit into DNA of candidate molecules.

Previous studies using space filling models indicated that3-phenylacetylamino-2,6 piperidinedione was capable of fully insertingbetween base pairs in DNA and forming a stereospecific hydrogen bondbetween the imino proton of the piperidinedione ring and a negativelycharged phosphate oxygen on the deoxyribose-phosphate backbone. Resultsemploying computer graphics confirmed this observation. Energycalculations further demonstrated that this compound had favorable vander Waals contacts of approximately -17.7 kcal when inserted into DNAwith an electrostatic energy of approximately -21.7 kcal resulting fromthe stereospecific hydrogen bond (2.7 Å) to phosphate. Increased fit ofthe ligand was obtained by substituting a para hydroxl group on thephenyl ring; this substitution enabled a second hydrogen bond to beformed between the hydroxyl group and a phosphate oxygen on the adjacentDNA strand. The increase in fit measured by energy calculations due tothe second hydrogen bond (2.64 Å) was reflected in an additional -24.6kcal in electrostatic energy. Other substitutions which were made on the3-phenylacetylamino-2, 6-piperidinedione skeleton did not significantlyincrease fit demonstrated by the normalized energy calculations forcertain halogenated analogs.

Synthesis

The synthesis of the unsubstituted derivative,3-N-phenylacetylamino-2,6-piperidinedione has been briefly described byBurzynski, et al., Drugs of the Future 10:103 (1985), and was used as ageneral method for the preparation of the desired compounds. Appropriatephenylacetic acids were reacted with N-hydroxysuccinimide in thepresence of N,N-dicyclohexylcarbodiimide (DCC) which gave succinimideesters. The active esters were stable enough to isolate for physical andspectroscopic characterization although the major portions of the esterswere used for the next reaction without purification. The active esterswere reacted with L-glutamine in the presence of sodium bicarbonate toobtain the glutamine derivatives. However, due to the difficulties ofobtaining the analytical samples, crude products were directly used forthe next reaction. To prepare active esters, the glutamine derivativeswere again reacted with N-hydroxysuccinimide in the presence of DCC togive the active esters which without purification were heated at95°-100° C. to obtain the desired 2,6-piperidinediones in variousyields. During the heating process the compounds were racemized.

Biological Evaluation

These synthetic derivatives were assessed for biological potency bymeasuring their growth inhibitory effects on various cell lines usingconcentrations of 4 nM based on the reported IC₅₀ of3-phenylacetylamino-2,6 piperidinedione in Nb2 cells. In YAK lymphomacells, the p-hydroxy compound was the most active derivative. Thiscompound, p-hydroxy-3-phenylacetylamino-2,6-piperidinedione was also themost active analog when tested in human leukemia (K652) cells. A doseresponse comparison in K562 cells showed that it was more active thanthe unsubstituted compound over the concentration range tested (10⁻⁵ to10⁻² M). Prolactin stimulated growth of rat Nb2 lymphoma cells wasinhibited by each of the compounds withp-hydroxy-3-phenylacetylamino-2,6-piperidinedione manifesting thegreatest activity. Compoundp-hydroxy-3-phenylacetylamino-2,6-piperidinedione was more active in Nb2lymphoma cells than 3-phenylacetylamino-2,6 piperidinedione over therange tested (10⁻⁴ M to 10⁻³ M).

Further analysis of growth inhibition of the most active analogp-hydroxy-3-phenylacetylamino-2,6-piperidinedione compared with theparent compound p-hydroxy-3-phenylacetylamino-2,6-piperidinedione wasperformed in MCF-7 (E-3) human breast cancer cells. Both3-phenylacetylamino-2,6 piperidinedione andp-hydroxy-3-phenylacetylamino-2,6-piperidinedione inhibited estrogensimulated cell growth. In a 9 day model,p-hydroxy-3-phenylacetylamino-2,6-piperidinedione was more active than3-phenylacetylamino-2,6 piperidinedione with IC₅₀ comparable totamoxifen (3-phenylacetylamino-2,6 piperidinedione, 3×10⁻³ M;p-hydroxy-3-phenylacetylamino-2,6-piperidinedione, 7×10⁻⁶ M, tamoxifen,1×10⁻⁷ M). The open chain hydrolysis product of 3-phenylacetylamino-2,6piperidinedione, PAG, did not inhibit cell growth even at highconcentrations (i.e., 10⁻² M).

Computer modeling coupled with energy calculations confirm that3-phenylacetylamino-2,6-piperidinedione is capable of inserting betweenbase pairs in partially unwound double stranded DNA and forming anenergetically favorable complex. A hydroxyl group placed in the paraposition of the phenyl ring of 3-phenylacetylamino-2,6-piperidinedioneenabled formation of a second hydrogen bond thereby linking both DNAstrands. This added hydrogen bond resulted in a greater fit in the DNAas assessed by energy calculations, i.e.,3-phenylacetylamino-2,6-piperidinedione (61%) versus the p-hydroxyderivative p-hydroxy-3-phenylacetylamino-2,6-piperidinedione (100%).Various substitutions at the para position as well as ortho and metapositions with fluorine and chlorine did not result in a significantincrease in fit compared to 3-phenylacetylamino-2,6-piperidinedione.

When the analogs of 3-phenylacetylamino-2,6-piperidinedione weresynthesized and examined for the capacity to inhibit cancer cell growth,the p-hydroxy derivative,p-hydroxy-3-phenylacetylamino-2,6-piperidinedione, which was predictedto be the most active compound based upon fit into DNA was consistentlyfound to be the most potent compound. That the capacity of the hydroxygroup of p-hydroxy-3-phenylacetylamino-2,6-piperidinedione to form asecond hydrogen bond to DNA was responsible for the predicted increasein activity is further supported by the lack of increased potency ofp-fluoro derivative as well as other halogenated derivatives which wereincapable of forming analogous hydrogen bonds. These observationsdemonstrate that among the compounds examined, a correlation existsbetween degree of fit into DNA and predicted biological potency. Thesefindings also support the contention that stereochemical complementarityof small molecules with nucleic acids can be a powerful tool fordesigning new drugs.

The mode of action of these piperidinediones is still not proven. Onemode of action might involve insertion into DNA as suggested by thecomputer modeling results and the observation that DNA synthesismeasured by thymidine incorporation was significantly inhibited upontreatment with 3-phenylacetylamino-2,6-piperidinedione in Nb2 lymphomacells. No covalent adducts of 3-phenylacetylamino-2,6-piperidinedionewith DNA were detected and the binding was observed to be weak andreversible, in comparison to classical intercalating drugs. Anotherpossible mode of action is suggested by the observation that compoundp-hydroxy-3-phenylacetylamino-2,6-piperidinedione inhibits estrogenstimulated cell growth in MCF-7 cells, which is comparable to that ofthe established anti-estrogen tamoxifen. In contrast to tamoxifen,however, neither 3-phenylacetylamino-2,6-piperidinedione norp-hydroxy-3-phenylacetylamino-2,6-piperidinedione exhibited appreciablebinding for the estrogen receptor. At the same time, direct binding ofsuch anti-estrogens to DNA appears to be weak. Taken as a whole, thesefindings support the possibility of a weak interaction of thepiperidinediones with both DNA and the estrogen receptor, involving areceptor mediated insertion of the ligand into DNA.

Development of Pharmacophores

Molecular modeling, as described above, facilitates the establishment ofthe best fit of molecules into nucleic acids such as double-stranded DNAbased on steric and electrostatic considerations. Individual molecules,such as estradiol, fit optimally into specific sites on DNA based on thelocation of specific nucleotides and the bonding characteristics ofindividual heteroatoms (see Example 2). Molecules that are related to aspecific molecule such as estradiol but display chemical differenceswill fit into the estradiol site with different degrees of precision:some may fit better and give rise to estradiol agonistic responses whilethose with poor fit display weak estrogenic activity. These differentmolecules may be aligned relative to the docking of heteroatoms withheteroatoms on the DNA to optimize electrostatic interactions. In thecreation of pharmacophores described below, molecules with activityequal to or greater than that of the hormone are chosen for alignment.To date, such molecules fit equally well or better than the hormone intoDNA using the energy calculation methodology described above. Moleculeswhich do not fit as well into DNA as the parent hormone are excludedfrom inclusion in the construction of the pharmacophore. The alignmentof the combined surfaces of the molecules occupies a specific volume ofspace thereby forming a three dimensional shape.

Pharmacophores are three dimensional arrangements of chemical groupsrelated to a given biological activity which enables meaningfulcomparison of molecules exhibiting the same biological function (Narutoet al., Eur. J. Med. Chem. 20:529-532 (1985)). Pharmacophores can bederived by simple overlap of active structures or common functionalgroups in the molecules. Without a way to orient the molecules e.g.,based upon fit with another macromolecule--a receptor, enzyme, or inthis case DNA, it is difficult and, in some cases, impossible toconstruct a reliable pharmacophore. This problem results in part fromthe fact that even closely related active molecules frequently fit intomacromolecules in very different ways.

A pharmacophore, as used herein, is defined as a 3-dimensional shapehaving a specific volume derived from the combined van der Waals surfaceof active molecules oriented by fit into DNA, coupled with point chargeslocated adjacent to the surface. A pharmacophore represents an aggregatearray of positions in space of a series of molecules having the same orsimilar biological activity. The van der Waals surface can berepresented in various ways including as a volume map, a dot surface, ora Connolly surface. The point charges are represented as dummy atomswhose positions are determined by the average positions of functionalgroups on active molecules which can form hydrogen bonds. Suitablecharges are placed on the dummy atoms consistent with the capacity ofthe active molecules to form hydrogen bonds. The pharmacophores arespecific for different compounds, their related molecules and aparticular biological activity. According to the present invention,within the general class of molecules called hormones, an estrogenpharmacophore, an anti-estrogen pharmacophore, an androgenpharmacophore, a thyroid hormone pharmacophore, and a toxicitypharmacophore (shown in FIGS. 2-5) have been disclosed. It should beemphasized that these created pharmacophores do not exist as such innature and are the product of aligning several related molecules tocommon binding sites in DNA using methods as described herein.

Many other pharmacophores have been constructed using the methoddescribed in this application. A pharmacophore, once created, standsalone and is subsequently independent from the nucleic acid that wasinvolved in its formation. Thus, after formation of the pharmacophore,one no longer needs to use the DNA as a template for the design ofbiologically active molecules. The pharmacophore itself can be used togenerate new molecules that will possess the same or similar structuraland charge features that are represented by the pharmacophore. This is acompletely different concept from the one of using the DNA as the modelfor the design of compounds. The pharmacophore can be used itself forany number of applications, including but not limited to the followingsas a screening tool for drug development; to determine if a particularcompound will possess bioactivity of a certain type, for instanceestrogenic or androgenic activity; for toxicological evaluation; and todesign compounds that possess increased or decreased binding affinityfor DNA.

Each pharmacophore has a characteristic shape, topology, volume, andelectrostatic profile. A pharmacophore is accurately described by itsthree dimensional shape which is represented by a coordinate system thatis configured in computer memory (see FIGS. 2-5 for examples ofpharmacophores. Each specific atom within a molecule that fits in apharmacophore has a specific location relative to the dockingheteroatoms. The individual atoms also have electrical charges assignedto them. These charges are represented numerically and through manyother ways including the use of colors and shading to indicate fieldstrength. As the degree of steric and electrostatic fit between thepharmacophore and the dummy atoms increases, resulting in a negativeenergy of interaction (-kcal), the efficacy of the pharmacophoreincreases which could manifest as increased bioactivity. The term"energy of interaction" as used herein is the total energy in -kcal of amolecule as it is being fitted into a pharmacophore. This has beenobserved in the case of molecules that fit within the estrogenpharmacophore and display bioactivity in a uterotropic assay. The volumeof a pharmacophore is described in cubic angstroms. The pharmacophorecan be cross sectioned precisely in any plane and internal distancesmeasured with an angstrom ruler. The circumference of any cross sectionis easily measured with morphometric analysis. Similarly, specificsubregions of the pharmacophore, such as the site that binds to the DNA,can be subjected to the same methods of analysis.

Construction and Utility of Pharmacophores

An example of the construction and utilization of the estrogenpharmacophore is given below. FIG. 2A is a computer generated spacefilling stereo view of the DNA cavity which fits estrogens. The fit ofactive estrogens oriented by energy calculations into the DNA cavity,using the methods described above, is presented in FIG. 2B. FIG. 2Cshows the combined active surface of estrogens removed from the cavityin DNA that is used to construct the pharmacophore. The atoms arecolored in the following manner: carbon/white; hydrogen/cyan;nitrogen/blue; oxygen/red; phosphorus/yellow. FIG. 3 demonstrates avolume contour map (yellow) in stereo with dummy atoms (magenta)surrounding the active molecules which were used in the construction ofthe pharmacophore (A); the empty pharmacophore (B); fit of the highlyactive estrogen 3,11β, 17β-Trihydroxy-7α-methylestra-1,3,5(10)-triene11-nitrate ester (hereinafter 7α-methylestradiol-11β-nitrate esterreported in Peters et al., J. Med. Chem. 32:2306-2310 (1989)) which isaccommodated completely within the pharmacophore (C); poor fit of theinactive estrogen 9β-estradiol which extends appreciably beyond thesurface of the pharmacophore (D). FIGS. 4 and 5 present examples of thethree dimensional appearance of estrogen, androgen, thyroid,anti-estrogen, and toxicity pharmacophores.

Quantitative measurements of the degree of fit of various compounds tothe pharmacophore are shown in FIG. 6. Fit is determined by measuringthe amount of volume of each structure which could be placed within thepharmacophore volume map and normalizing the value to that of thenatural hormone estradiol set at 50%. Electrostatic interactions withthe dummy atoms are optimized for each compound and calculated using theTripos force field. The electrostatic energy value is normalized to thatof estradiol set at 50%. In this study, the total of volume fit andelectrostatic fit are treated equally and totaled to reflect the overallfit in the pharmacophore. As shown in FIG. 6, degree of fit to thepharmacophore correlates highly to relative uterotropic (estrogenic)activity. In comparison to estradiol, 7α-methylestradiol-11β-nitrateester (labeled 2), which is not part of the data set used to constructthe pharmacophore, fits appreciably better than estradiol (labeled 1).In contrast, 7α-methylestradiol-11β-nitrate ester binds poorly to theestrogen receptor (less than 6% of the binding of estradiol). Theuterotropic values for 7α-methylestradiol-11β-nitrate ester (labeled 2)relative to estradiol set to a normalized value of 100% are considerablygreater than that of estradiol (Peters et al., J. Med. Chem.32:2306-2310 (1989)) as predicted by fit into the pharmacophore. Incontrast, it is not possible to fit 9β-estradiol (labeled 3) into thepharmacophore and, as predicted, this analog has little uterotropicactivity. In summary, fit of compounds to the pharmacophore correlateswith biological activity and can thus be used to design new compounds byvirtue of their fit. It is noteworthy that the highly potent estrogenicnitrate ester binds very poorly to the estrogen receptor. Thus, it wouldnot be possible to predict the estrogenic activity of this analog on thebasis of receptor binding or from a pharmacophore derived from aputative binding site in a protein receptor. In many cases, compoundswith greater estrogenic activity than the natural hormone estradiol bindrelatively poorly to the estrogen receptor.

The fit of compounds into DNA is consistent with, but not the same as,fit into the pharmacophores. The distinction between the DNA cavitiesand the pharmacophores is that the surfaces were derived from differentstructures, i.e. the DNA cavities from the DNA structure and thepharmacophores from the combined surfaces of active compounds. As shownin the examples and in FIGS. 2-5, the degree of fit to thepharmacophores is obtained by fit to the three dimensional map whichrepresents the active compounds. The pharmacophores enable quantitativedetermination of the degree of fit to the combined surfaces of theactive compounds and this information cannot be obtained from fit intoDNA. Moreover, the fit of active compounds to the pharmacophore can bequantitated based upon the portion of the molecule which does not fitwithin the pharmacophore volume. This enables automatic assessment ofpredicted inactive structures.

Toxicity Pharmacophores (Toxicophores)

Pharmacophores can be constructed to represent a three dimensional shapethat is predictive of toxic biological activity. Such pharmacophores,called toxicophores, have regions that would potentially damage DNA. Atypical toxicophore has been constructed using, tetrodotoxin, dioxin,RU486, dilantin, thalidomide and oroflex, among other compounds. Anexample of this toxicophore is provided in FIGS. 4E and 5E. For example,by overlaying this toxicophore on another pharmacophore such as theestrogen phartnacophore (FIGS. 4A and 5A), a drug designer would know toavoid designing an estrogenic compound with certain molecular groupsthat might impart toxic activity if these groups extended into the threedimensional space occupied by the toxicophore. This approach wouldgreatly facilitate and economize drug design by guiding the designer toavoid synthesizing estrogenic compounds that might have damaging effectson DNA as opposed to proceeding with synthesis and purification andsubsequently discovering that the compound possesses dangerous toxicity.

Solvent Pharmacophores (Aquaphores)

Pharmacophores in their relationship to nucleic acids are usuallysurrounded by a solvent. The predominant solvent in living organisms iswater and accordingly, most pharmacophores exist in an aqueousenvironment. Water is the preferred embodiment of the solventpharmacophore and is termed an aquaphore. Pharmacophores, and theirmolecules may also be placed in non-aqueous environments for variouspurposes such as crystallographic studies or other analyticalprocedures.

In living organisms, the aqueous environment surrounding thepharmacophore also has an intimate association with the adjacent nucleicacid. This aqueous shell assists in the optimal fit of the pharmacophoreinto the cavity of the double stranded DNA, and has its own threedimensional shape. The optimal steric and electrostatic placement ofwater molecules in the space between the pharmacophore and the DNA isachieved in the present invention. This three dimensional shape iscalled a solvent pharmacophore, and can be described in all the wayslisted above for the pharmacophores based on other molecules such asestrogen. Solvent pharmacophores assist the designer of compounds byplacing limits on the dimensions of a compound designed using aparticular pharmacophore as a template. In addition, solventpharmacophores assist the creator of pharmacophores because the solventshell or cage represented by the pharmacophore provides enhanced abilityto properly align molecules relative to DNA during the creation of thepharmacophore.

Receptophore

Many molecules, such as steroid hormones, are shuttled to the nucleus byother molecules known as receptors (Tsai and O'Malley, Ann. Rev.Biochem. 63:451-486 (1994)). These receptors bind the hormones (calledligands), bind to the nucleic acids, for example in their DNA bindingdomain, and present ligands to nucleic acids such as DNA. Evidencesuggests that the binding of the receptor to the DNA causes aconformational change in the DNA to facilitate insertion of the ligand(Nardulli et al., Molec. Endocr. 7:331-340 (1993)). The pharmacophoreconcept is based on the three dimensional shape of the optimal fit ofrelated molecules into nucleic acids such as partially unwound,double-stranded DNA. The DNA binding domain of the receptor can bemodeled into a three dimensional shape based on the same principlesdescribed above for the pharmacophore. The resultant shape is termed areceptophore and is the three dimensional representation of the sites ofinteraction of the receptor and the nucleotides of the DNA. The DNAbinding region of each receptor likely gives rise to a differentreceptophore. This receptophore provides a valuable tool to moleculardesigners interested in developing new receptors, or in modulatingreceptor binding to DNA.

Receptophore-Pharmacophore Pairs

The nucleic acid binding region of receptors and their ligands can bemodeled as receptophores and pharmacophores, respectively. Theconfiguration of the receptophore and its associated pharmacophore intheir proper alignment relative to their respective DNA binding regionsconstitutes a specific pair of shapes that represents the minimalmolecular unit for DNA binding and ligand insertion. Designers ofcompounds utilize this information to synthesize and screen molecules tomodify the facility of docking and ligand insertion. Such modificationsmay provide a host of new therapies such as treatments for hormonedependent carcinomas of the prostate or breast.

Metabophores

Most naturally occurring compounds are derived from antecedents orprecursors in a synthetic pathway and also are destined for inactivationin a catabolic pathway. Many precursors and metabolites of compounds areless active due to the addition of an extra group such as a methyl groupor acetylation of a specific site. In some cases, precursors andmetabolites of a molecule have different groups added sequentially tothe same site on the active molecule, creating a side chain. Knowledgeof a site of preferred addition or deletion of chemical groups assistsin the design of molecule with enhanced or reduced activity.

These sites can be modeled relative to the pharmacophore to produce athree dimensional representation of a preferred site for modification ofthe molecule. This three dimensional representation, termed ametabophore, provides constraints for rational design of active andinactive variants of the parent molecules that fit into thepharmacophore. Analysis of which chemicals can effectively be added atthe attachment point of the metabophore to the pharmacophore reveals themost favorable molecules to pursue for synthesis, purification andtesting.

It will be appreciated that other embodiments and uses will be apparentto those skilled in the art and that the invention is not limited tothese specific illustrative examples.

EXAMPLE 1 Fit Into Partially Unwound Double Stranded DNA Using theMammalian Female Hormone Estradiol

Computer modeling has demonstrated that the mammalian steroidprogesterone is a remarkable "lock and key" fit into DNA at 5'-dTdG-3',5'-dCdA-3' (FIG. 1). Each of the known x-ray crystal structures ofprogesterone is capable of forming two stereospecific hydrogen bonds anda stable complex measured by force-field calculations. Remarkablecomplementarity is evident in the complex by the overlap of hydrophilicand hydrophobic regions of the steroid and DNA. The enantiomer ofprogesterone which does not occur in nature does not fit. The planthormone gibberellic acid has also been shown to fit between base pairsbut in a different sequence, i.e., 5'-TdA-3', 5'-dTdA-3'. Fourstereospecific hydrogen bonds are formed within the couples:ent-gibberellic acid does not fit.

The mammalian female hormone estradiol also fits in DNA (FIG. 1). Twostereospecific hydrogen bonds of approximately 2.65 Å are formed betweeneach hydroxyl group of the steroid and phosphate oxygens on adjacentstrands. The overall fit within the complex is about -59 kcal. Mostalterations of the positions of the hydroxyl groups on the estratrienenucleus result in a substantial loss of potential electrostaticinteractions with the DNA. Moreover, most alterations of the absolutestereochemistry of the cyclopentanophenanthrene ring pattern also resultin a substantial loss of potential electrostatic interactions with theDNA. Moreover, most alterations of the absolute stereochemistry of thecyclopentanophenanthrene ring pattern also result in a poor fittingmolecule. This is evident in attempting to fit 9β-estradiol into DNA.The puckering in the steroid caused by inversion of the estradiolstereochemistry at C-9 from α to β prevents complete insertion betweenthe base pairs. Even if forced into DNA without regard to strain causedby the overlap of van der Waals surfaces, 9β-estradiol can form only asingle hydrogen bond. The relative fit into DNA resulting from partialinsertion of 9β-estradiol is about -17 kcal 9β-estradiol is inactivewhen tested in vivo for estrogenic (uterotropic) activity.

The finding that estradiol is a "lock and key" fit into DNA, althoughmost structural alternatives to estradiol fit poorly provides furthersupport for the premise that DNA stereochemistry contains the masterblueprint for natural product structures.

EXAMPLE 2 Correlation of Estrogenic Activity with the Fit of Estrogensand Related Analogs into DNA

That fit into DNA measured by energy calculations can be correlated withbiologic activity was demonstrated using a series of estrogens andrelated synthetic analogs. The molecules which are inactive inuterotropic assays fit poorly into DNA. Molecules that fit into DNAbetter than estradiol are all more active than estradiol in theuterotropic assays, for example, 11β-acetoxyestradiol (approximately -68kcal). This correlation is also observed with nonsteroidal, syntheticestrogens, such as the potent synthetic estrogen,transdiethylstilbestrol (approximately -62 kcal), which fits well,whereas the poorly active geometrical isomer cisdiethylstilbestrol(approximately -20 kcal) is a poor fit.

EXAMPLE 3 Biosynthetic Pathways Reflect Increasing Fit into DNA WhereasInactivation Pathways Lead to Decreasing Fit into DNA

Molecular modeling studies conducted with the mammalian hormoneprogesterone and the plant hormone gibberellic acid have shown that eachstep in the respective biosynthetic pathway reflects a structural changethat results in increased fit in DNA. For example, when consideringpossible stereoisomers which could result in any given step inprogesterone biosynthesis, the best fitting structure is one which wasproduced in nature. In sharp contrast, each step in the inactivation ofprogesterone eventually leading to the excreted glucuronides andsulfates resulted in the worst possible fitting stereoisomers.

The two possible dihdro reduced metabolites of the male hormonetestosterone were examined for fit and correlation with biologicalactivity. Relative to testosterone (100%), 5β-dihydrotestosterone is apoor fit (84%), whereas its epimer 5α-dihydrotestosterone fits evenbetter than testosterone (102%). These data are consistent withpublished findings by Hilgar and Hummel, "The androgenic and myogenicevaluation of steroids and other compounds -assay 1", in A G Hilgard. D.J. Hummel (ed.) Endocrine Bioassay Data, Part III)U.S. Dept. HEW NIH1964), that 5α-dihydrotestosterone is a highly active androgen, whereas5β-dihydrotestosterone is essentially inactive.

In the case of the tetrahydro reduced stereoisomers of progesterone,3α-hydroxy-5β-pregan-20-one was a poor fit whereas its stereoisomer3α-hydroxy-5α-pregnan-20-one was an excellent fit. The former moleculeis a highly active neurosteroid, as reported by Purdy, et al., J. Med.Chem. 33:1572-1581 (1990). Comparison of the specific pattern ofdonor/acceptor linkages of 3α-hydroxy-5α-pregan-20-one with those in thesteroid/thyroid hormone superfamily demonstrated that the linkagepattern of 3α-hydroxy-5α-pregan-20-one is unique. Compounds having suchunique linkages are predicted to have unique biologic function and maybe ligands for newly discovered "orphan receptors."

EXAMPLE 4 Correlation of Toxicity and "Side Effects" with Fit into DNA

Ligands that fit into more than one site in DNA have been observed tohave multiple biologic actions. Both desirable and undesirable "sideeffects" should thus be predictable from the specific DNA sequence whicha given compound fits into as well as the manner and relative degree offit. Examples of molecules that have been observed to fit into more thanone site in DNA include the psychotropics cocaine, morphine, LSD andtetrahydrocannabinoids, and certain intercalating antibiotics. Forexample, the monoamine oxidase inhibitor selegiline fits into the sitein DNA which accommodates glucose and various oral antidiabetic drugs asreported by Rowland et al., J. Clin. Pharmacol. 34:80-85(1994). Thisobservation is consistent with the finding that selegiline causeshypoglycemia in some patients. Another case is the anti-androgenanandron which fits into the site in DNA which accommodatestestosterone. Because anandron fits into DNA in two orientations, i.e.,in a manner similar to both androgens and anti-androgens, mixed activityis predicted for this compound. Experimental results indicate thatanandron has both agonist and antagonist activities as reported bySteinsapir, et al., The Endocrine Society (74th Annual Meeting) 1992:109(abs. 228).

It has also been noted that ligands which cause stress, chemicalmodifications and/or covalent linkages to the DNA when fit into a givensite frequently possess toxicity. Examples include certain carcinogensand teratogens, e.g., thalidomide, dioxin, arene oxides, aflatoxins andsome diethylstilbestrol metabolites. Another example is theanti-progestin RU486 which stresses base pair hydrogen bonds wheninserted fully into the progesterone site in DNA. Similar strain isproduced by other anti-progestins having the same side chains (e.g.,11β-phenylamines) raising the possibility that such features maycorrelate with abortifacient activity attributed to RU486 and relatedanalogs. Observations with thalidomide enantiomers indicate thatteratogenicity associated with this compound may correlate with astereospecific effect on base pairing. An example of such a toxicitypharmacophore, called a toxicophore, is presented in FIGS. 4E and 5E andthe corresponding data file is submitted on magnetic tape.

EXAMPLE 5 Design and Development of New Drugs

Using the principles described above and in the examples, new drugs canbe designed or existing drugs can be redesigned while at the same timelimiting potential undesirable side effects. One example of how anactive drug can be designed using the technology follows:

3-Phenylacetylamino-2,6-piperidinedione (A10) is a modified amino-acidderivative, which was originally isolated from freeze-dried human urine.Despite having low toxicity, high concentrations of A10 were required todemonstrate significant growth inhibitory activity on tumor cells. Thefocus of the following study was to develop more potent analogs.Modeling studies demonstrated that A10 was capable of inserting intopartially unwound double stranded DNA and forming a single hydrogen bondbetween the imino proton of the piperidinedione ring and a phosphateoxygen on a single strand. It was observed that placing a hydroxyl groupat the para position of the phenyl group of A10 would enable a secondhydrogen bond to form thereby substantially enhancing fit as reported byHendry et al., J. Steroid Biochem. Molec. Biol. 48:495-505 (1994), theteachings of which are hereby incorporated by reference in theirentirety. The relative fit of A10 (normalized to 100%) and variousrelated analogs measured by energy calculations demonstrate that thebest fitting compound is p-OH-A10 (164%). Subsequent synthesis of thesecompounds followed by testing in various animal and human tumor cellsdemonstrated that p-OH-A10 was the most active compound and was as muchas an order of magnitude more active than A10, as reported by Hendry, etal., Recent Advances in Chemotherapy, Buchner and Rubinstein (eds)2498-2499 (1991), Hendry et al., U.S. Pat. No. 5,238,947 which isincorporated herein by reference.

EXAMPLE 6 Comparison of the Drug Design Technology to ClasicalStructure-Activity Methods

The drug design technology described here can be used in conjunctionwith quantitative-structure-activity-relationship methods (QSAR), e.g.,comparative field molecular analysis (CoMFA). One value of the approachis that it facilitats the orientation of various ligands relative to oneanother in three dimensions. The successful structure-activityrelationship found for estrogens derived from fit into DNA is describedhere. If one were to attempt to derive such a relationship a prioriwithout first knowing the detailed three-dimensional structure of anappropriate macromolecule (e.g., the ligand binding site of a receptoror an enzymatic site), chemical intuition would necessitate searchingfor common features that exist in known active structures. In the caseof the natural hormone estradiol and the potent synthetic estrogentrans-diethylstilbestrol, such a common feature is the phenoxy group.Alterations of the phenoxy group give rise to inactive structures. Ifone overlaps the resulting three dimensional orientation with that whichis obtained by optimal docking of these molecules into DNA, a differentpattern emerges. Thus, one would expect that such different orientationswhen used subsequently to correlate activity of other molecules wouldgive very different results. In fact, analysis of relative fit into DNAin kcal shows that if the orientation of trans-diethylstilbestrol basedupon overlapping the A ring of estradiol is used to dock the ligand intoDNA, a relatively poor fit results. In this case, poor activity fortrans-diethylstilbestrol would be incorrectly predicted. In contrast,using the orientation of DES derived only from the stereochemistry ofDNA as taught by the present invention, increased activity would becorrectly predicted.

EXAMPLE 7 Design of an Anti-Estrogen,Para-Hydroxyphenylacetylamino-2,6-Piperidinedione, a Regulator of YumorCell Growth

Within the past few years there has been a growing interest in nontoxic,naturally occurring small molecules as regulators of tumor cell growth.Examples of recently published findings include: regression of mammarycarcinomas by a dietary monocyclic nonoterpens, limonene; modulation ofoncogene expression in erythroleukemic cells growth by an endogenousproduct of lipid peroxidation, 4-hydroxynonenal; inhibition of malignantcell growth by the endogenous ligand p-hydroxyphenylacetate; andinduction of tumor cell differentiation in premalignant and malignantcells by a circulating component of human plasma, phenylacetate.

Phenylacetate has been shown to reduce levels of the myc oncogene whichis involved in the development of several cancers including breast,brain, prostate, blood, lung and colon. Another mechanism by whichphenylacetate is thought to be effective is by reducing levels of theamino acid glutamine. Phenylacetate conjugates with circulatingglutamine to produce the excreted urinary metabolitephenylacetylglutamine (PAG). Cancer cells require glutamine for growthand are known to be more sensitive to glutamine depletion that normalcells. These findings have led to the initiation of Phase I clinicaltrials with phenylacetate in brain and prostate cancer at the NationalCancer Institute.

In the process of screening fractions of freeze dried human urine forgrowth inhibitory activity in human breast cancer cells, a dehydrationproduct of PAG was isolated. The compound was characterized as3-phenylacetylamino-2,6-piperidinedione by spectroscopic methods andindependent synthesis and was termed A10 based upon the chromatographicfraction from which it was isolated. It has not been conclusivelydetermined whether this structure is a circulating compound, however, itis similir to phenylacetate in that it lacks toxicity in both laboratoryanimals and humans. The compound has also been found to inhibit growthin a variety of cancer cells in vitro, as well as human breast cancertransplanted into athymic nude mice. Chemoprevention effects have alsobeen reported. In addition to phenylacetate and methylp-hydroxyphenylacetate, there is a number of synthetic compounds withantitumor activities that have structural features in common with3-phenylacetylamino-2,6-piperidinedione. Examples of such compounds inwhich analogies to the piperidinedione ring are prominent include thearomatase inhibitors aminoglutethimide, rogletimide and related analogswhich are used in the treatment of breast cancer; the alkylating drugPCNU which inhibits tumor growth by proposed interaction with DNA;5-cinnamoyl-6-aminouracil derivatives which inhibit tumor growth viaputative DNA intercalation; amonafide and its congeners which mediatetopoisomerase II DNA cleavage by intercalation, bis(2,6-dioxopiperazine) derivatives, e.g., ICRF-193, which are potent,direct inhibitors of mammalian DNA topoisomerase II.

Relative high doses of 3-phenylacetylamino-2,6-piperidinedione have beengenerally required to inhibit tumor growth both in vitro and in vivo.The goal of this study was to identify more activephenylacetylamino-2,6-piperidinediones using the technique describedabove. The technology is based upon modeling of the stereospecific fitof molecules into DNA and has been recently modified to take advantageof computer graphics and energy calculations. Computer modeling wasfollowed by the design, synthesis, and in vitro biological testing of3-phenylacetylamino-2,6-piperidinedione derivatives. The moleculepredicated to be the most active based upon degree of fit in DNA, i.e.,the p-hydroxy derivative, was found to be the most active antitumoragent in all of the biological assays investigated. When tested in MCF-7(E3) human breast cancer cells, the p-hydroxy derivative possessedantiestrogenic activity in the range of the drug tamoxifen which iscurrently in clinical use for the treatment of breast cancer.

EXAMPLE 8

Three separate molecules designed with modeling technology are shown inFIG. 7. These molecules, termed SGI 100, SGI 101 and SGI 102 bearsimilarities and differences to each other. They all show structuralsimilarities to components of both estrogen and progesterone. SGI 100was designed on the basis of its ability to fit into the site in DNAwhich accommodates both estradiol and progesterone. The manner in whichit fits predicted antagonist activity. When fit to the estrogenpharmacophore (FIG. 2), the acetyl group at the 17β position extendedout of the pharmacophore and had electrostatic repulsion with dummyatoms (-131 kcal) compared to estrogen (-51 kcal). Accordingly, thishigh positive energy of interaction indicates that SGI 100 acts as anantagonist. The binding of SGI 100 to the estrogen receptor is dosedependent and approximately 144 times less than estradiol. Inbioactivity experiments, SGI 100 significantly decreased cell growth inMCF-7 human breast cancer cells (134,431 cells) at a dose of 10⁻⁸ M whencompared to control cells (252,197 cells). The same concentration oftamoxifen citrate decreased the number of MCF-7 cells to 187,759. Thus,the design of this compound based on the pharmacophore approach of thisinvention predicted a demonstrable anti-estrogen bioactivity that wasgreater than tamoxifen. SGI 100 binds in a dose dependent manner to theprogesterone receptor but with 133 to 200 times less affinity.

SGI 101 was designed on the basis of fitting into DNA at the site whichaccommodates estradiol but with opposite hydrogen bonding propertieswhich predict estrogen antagonist activity. SGI 101 extends beyond theestrogen pharmacophore and has electrostatic repulsion between thepara-nitro group and dummy atoms. SGI 101 is the most potent analogdesigned by the technology as measured by growth inhibition of MCF-7cells. At a dose of 10⁻⁸ M, SGI 101 inhibited cell growth (81,103 cells)relative to control (252,197 cells) and was substantially more activethan the same concentration of tamoxifen (187,759 cells).

SGI 102 was designed on the basis of its fit into DNA at the site whichaccommodates progesterone. SGI 102 possesses an alkyl amino side chainat the 11β position which extends out of the site between base pairsinto the major groove. It has different hydrogen bonding properties thanprogesterone and would extend beyond the volume map of the progesteronepharmacophore. As such, it is predicted to be an antagonist. SGI 102 wasdesigned prospectively, synthesized, and tested in various biologicalassays. SGI 102 binds in a dose dependent manner to the progesteronereceptor but not as strongly as progesterone or the abortifacientantiprogestin RU486. In animal experiments, SGI 102 showed noabortifacient activity. However, in experiments using MCF-7 human breastcancer cells, SGI 102 had equivalent activity to RU486 in inhibitinggrowth. These findings are consistent with the predictions made by themodeling technology.

It should be understood that the foregoing relates only to a preferredembodiment of the present invention and that numerous modifications oralterations may be made therein without departing from the spirit andthe scope of the invention as set forth in the appended claims.

I claim:
 1. A computer-based method for comparing energies ofinteraction of two molecules with a nucleic acid, the method be usefulin designing a molecule with a desired biological activity, comprisingthe computer implemented steps of:choosing a first molecule with thedesired biological activity; determining a site comprising a nucleicacid sequence which accommodates the first molecule; calculating anenergy of interaction between the first molecule and the site; designinga second molecule which fits into the site; and calculating an energy ofinteraction between the second molecule and the site to determine if thesecond molecule has an equal or lower energy of interaction than thefirst molecule for the site, wherein the second molecule has the desiredbiological activity if the energy of interaction of the second moleculefor the site is equal to or lower than the energy of interaction of thefirst molecule for the site.
 2. The computer-based method of claim 1wherein the nucleic acid is selected from the group consisting ofdeoxyribonucleic acid, double stranded deoxyribonucleic acid,ent-deoxyribonucleic acid, ribonucleic acid, double stranded ribonucleicacid, complexes of deoxyribonucleic acid and ribonucleic acid, complexesof ribonucleic acid and ribonucleic acid, and apurinic and apyrimidinicsites.
 3. The computer-based method of claim 1 wherein the nucleic acidis deoxyribonucleic acid.
 4. The computer-based method of claim 2wherein the nucleic acid is double stranded deoxyribonucleic acid andthe sequence is 5'-dTdG-3', 5'-dCdA-3'.
 5. The computer-based method ofclaim 2 wherein the nucleic acid is double stranded deoxyribonucleicacid and the sequence is 5'-dTdA-3', 5'-dTdA-3'.
 6. The computer-basedmethod of claim 2 wherein the double stranded deoxyribonucleic acid ispartially unwound.
 7. The computer-based method of claim 1 wherein thebiological activity is hormonal, neurotransmitter, metabolic, genetic,immunologic, pathologic, toxic, anti-diabetic, or anti-mitotic.
 8. Thecomputer-based method of claim 7 wherein the biological activity ishormonal and the hormonal activity is estrogenic, anti-estrogenic,androgenic, anti-androgenic, progestational, anti-progestational,mineralocorticoid, retinoid, vitamin D-like, thyroid, ecdysone-like,gibberellic acid-like, or glucocorticoid hormonal activity.
 9. Thecomputer-based method of claim 1 wherein the first molecule has acyclopentanophenanthrene motif.
 10. The computer-based method of claim 1wherein the second molecule has a cyclopentanophenanthrene motif. 11.The computer-based method of claim 1 wherein the site in the nucleicacid which accommodates the first molecule and the second molecule isdetermined by the computer-implemented method further comprising:using astereoviewer for docking the first molecule and the second molecule inthe site; and using van der Waals dot, mesh, and space filling surfacesto guide the docking of the molecules and to minimize steric strain ofthe nucleic acid.
 12. The computer-based method of claim 1 wherein thecalculation of the energy of interaction between the first molecule andthe site comprises the computer-implemented steps of:calculating a valuefor steric fit by optimizing van der Waals interactions of the firstmolecule with the nucleic acid; calculating a value for hydrogen bondingfit by measuring a change in electrostatic energy using charges on donorhydrogens and acceptor heteroatoms; and adding the calculated values.13. The computer-based method of claim 1 wherein the calculation of theenergy of interaction between the second molecule and the site comprisesthe computer-implemented steps of:calculating a value for steric fit byoptimizing van der Waals interactions of the second molecule with thenucleic acid; calculating a value for hydrogen bonding fit by measuringa change in electrostatic energy using charges on donor hydrogens andacceptor heteroatoms; and adding the calculated values.
 14. Acomputer-based method for comparing energies of interaction of twomolecules with a nucleic acid the method being useful in predicting abiological activity of a molecule, comprising the computer implementedsteps of:choosing a first molecule with the biological activity;determining a site comprising a nucleic acid sequence which accommodatesthe first molecule; calculating an energy of interaction between thefirst molecule and the site; determining whether a second molecule fitsinto the site; and calculating an energy of interaction between thesecond molecule and the site to determine if the second molecule has anequal or lower energy of interaction than the first molecule for thesite, wherein the second molecule has the desired biological activity itthe energy of interaction of the second molecule for the site is equalto or lower than the energy of interaction of the first molecule for thesite.
 15. The computer-based method of claim 14 wherein the nucleic acidis selected from the group consisting of deoxyribonucleic acid, doublestranded deoxyribonucleic acid, ent-deoxyribonucleic acid, ribonucleicacid, double stranded ribonucleic acid, complexes of deoxyribonucleicacid and ribonucleic acid, complexes of ribonucleic acid and ribonucleicacid, and apurinic and apyrimidinic sites.
 16. The computer-based methodof claim 15 wherein the nucleic acid is deoxyribonucleic acid.
 17. Thecomputer-based method of claim 15 wherein the nucleic acid is doublestranded deoxyribonucleic acid and the sequence is 5'-dTdG-3',5'-dCdA-3'.
 18. The computer-based method of claim 15 wherein thenucleic acid is double stranded deoxyribonucleic acid and the sequenceis 5'-dTdA-3', 5'-dTdA-3'.
 19. The computer-based method of claim 15wherein the double stranded deoxyribonucleic acid is partially unwound.20. The computer-based method of claim 14 wherein the biologicalactivity is hormonal, neurotransmitter, metabolic, genetic, immunologic,pathologic, toxic, anti-diabetic, or anti-mitotic.
 21. Thecomputer-based method of claim 20 wherein the biological activity ishormonal and the hormonal activity is estrogenic, anti-estrogenic,androgenic, anti-androgenic, progestational, anti-progestational,mineralocorticoid, retinoid, vitamin D-like, thyroid, ecdysone-like,gibberellic acid-like, or glucocorticoid hormonal activity.
 22. Thecomputer-based method of claim 14 wherein the first molecule has acyclopentanophenanthrene motif.
 23. The computer-based method of claim14 wherein the second molecule has a cyclopentanophenanthrene motif. 24.The computer-based method of claim 14 wherein the site in the nucleicacid which accommodates the first molecule and the second molecule isdetermined by docking the first molecule and the second molecule in thesite comprising use of van der Waals dot, mesh, and space fillingsurfaces to guide the docking of the first molecule and the secondmolecule and to minimize steric strain of the nucleic acid.
 25. Thecomputer-based method of claim 14 wherein the site in the nucleic acidwhich accommodates the first molecule and the second molecule isdetermined by a method further comprising the computer-implemented stepsof:using a stereoviewer for docking the molecules in the site; and usingvan der Waals dot, mesh, and space filling surfaces to guide the dockingof the molecules and to minimize steric strain of the nucleic acid. 26.The computer-based method of claim 14 wherein the calculation of theenergy of interaction between the first molecule and the site comprisesthe computer-implemented steps of:calculating a value for steric fit byoptimizing van der Waals interactions of the molecule with the, nucleicacid; calculating a value for hydrogen bonding fit by measuring a changein electrostatic energy using charges on donor hydrogens and acceptorheteroatoms; and adding the calculated values.
 27. The computer-basedmethod of claim 14 wherein the calculation of the energy of interactionbetween the second molecule and the site comprises thecomputer-implemented steps of:calculating a value for steric fit byoptimizing van der Waals interactions of the molecule with the nucleicacid; calculating a value for hydrogen bonding fit by measuring a changein electrostatic energy using charges on donor hydrogens and acceptorheteroatoms; and adding the calculated values.
 28. The method of claim1, further comprising the step of synthesizing the second molecule. 29.A molecule made with the method of claim 28 having the structure of:##STR1##
 30. A molecule made with the method of claim 28 having thestructure of: ##STR2##
 31. A molecule made with the method of claim 28having the structure of: ##STR3##